Mitochondrial Proteostasis of Cytosolic TDP-43 Protein Aggregates Mediates Mitochondrial Dysfunction in C9orf72 ALS
Introduction. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons.1 About 90% of diagnosed patients die within 5 years of onset.1 Currently, two medications have been approved for ALS treatment – riluzole and edaravone.1,2 Riluzole increases survival only by about 3 months, and edaravone is targeted specifically to ALS patients of early onset and rapid progression.1,2 The most commonly implicated genetic cause of ALS is a GGGGCC (G4C2) repeat in the C9orf72 gene, which induces cytosolic TDP-43 aggregates.3,4 TDP-43 aggregates are then imported into the mitochondria, leading to mitochondrial dysfunction and neuronal death.5,6 Lon protease (LonP1) typically degrades damaged proteins in the mitochondria as part of mitochondrial proteostasis.7 Additionally, LonP1 has been shown to contribute to degradation of cytosolic protein aggregates in yeast cells.8 The same mechanism may extend to human cells. The role of mitochondrial proteostasis in the import of cytosolic TDP-43 aggregates suggests a potential novel therapy for ALS. Methods. Tetracycline (Tet) inducible HEK293 cell lines expressing TDP-43 were used to prepare cell lysates.9 Coimmunoprecipitation assay with anti-TDP-43 antibody was used to determine TDP-43 interaction with LonP1.9 Immuno-electron microscopy was used to observe the mitochondria.9 Cell lysates were transfected with LonP1-expressing plasmid and induced by Tet to determine the effect of increased LonP1 expression.9 Cell lysates were transduced with a lentivirus expressing shRNA against LonP1 to determine the effect of decreasing LonP1 expression.9 Fractionation experiments were used to isolate mitochondrial and cytoplasmic protein.9 Western blotting was used to quantify protein expression.9 To examine in vivo impact of decreased LonP1, TDP-43 transgenic flies were crossed with flies expressing siRNA against LonP1.9 TDP-43 expression was induced by heat shock.9 Quantitative analysis was used to determine mitochondrial damage.9 Motor function was measured under the Elav-Gal4/Tub-Gal80ts driver.9 Results. TDP-43 interacts with LonP1 within the mitochondria.9 Increased LonP1 expression resulted in decreased TDP-43 protein within the mitochondria and decreased cell cytotoxicity.9 Decreased LonP1 expression resulted in increased TDP-43 protein within the mitochondria and increased cell cytotoxicity.9 Decreased LonP1 in vivo resulted in increased mitochondrial TDP-43 and damage as well as faster progression of locomotor dysfunction.9 Conclusion. Cytosolic TDP-43 aggregates are imported into the mitochondria, where LonP1 can degrade the TDP-43 aggregates. In doing so, LonP1 plays a protective role against mitochondrial damage and locomotor deficits. Therefore, increasing LonP1 expression may be a potential therapy for ALS.
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